Genome-wide association studies using multi-models and multi-SNP datasets provide new insights into pasmo resistance in flax.

Introduction: Flax (Linum usitatissimum L.) is an economically important crop due to its oil and fiber. However, it is prone to various diseases, including pasmo caused by the fungus Septoria linicola. Methods: In this study, we conducted field evaluations of 445 flax accessions over a five-year period (2012-2016) to assess their resistance to pasmo A total of 246,035 single nucleotide polymorphisms (SNPs) were used for genetic analysis. Four statistical models, including the single-locus model GEMMA and the multi-locus models FarmCPU, mrMLM, and 3VmrMLM, were assessed to identify quantitative trait nucleotides (QTNs) associated with pasmo resistance. Results: We identified 372 significant QTNs or 132 tag QTNs associated with pasmo resistance from five pasmo resistance datasets (PAS2012-PAS2016 and the 5-year average, namely PASmean) and three genotypic datasets (the all SNPs/ALL, the gene-based SNPs/GB and the RGA-based SNPs/RGAB). The tag QTNs had R2 values of 0.66-16.98% from the ALL SNP dataset, 0.68-20.54%from the GB SNP dataset, and 0.52-22.42% from the RGAB SNP dataset. Of these tag QTNs, 93 were novel. Additionally, 37 resistance gene analogs (RGAs)co-localizing with 39 tag QTNs were considered as potential candidates for controlling pasmo resistance in flax and 50 QTN-by-environment interactions(QEIs) were identified to account for genes by environmental interactions. Nine RGAs were predicted as candidate genes for ten QEIs. Discussion: Our results suggest that pasmo resistance in flax is polygenic and potentially influenced by environmental factors. The identified QTNs provide potential targets for improving pasmo resistance in flax breeding programs. This study sheds light on the genetic basis of pasmo resistance and highlights the importance of considering both genetic and environmental factors in breeding programs for flax.

Insights into the Genetic Architecture and Genomic Prediction of Powdery Mildew Resistance in Flax (Linum usitatissimum L.).

Powdery mildew (PM), caused by the fungus Oidium lini in flax, can cause defoliation and reduce seed yield and quality. To date, one major dominant gene (Pm1) and three quantitative trait loci (QTL) on chromosomes 1, 7 and 9 have been reported for PM resistance. To fully dissect the genetic architecture of PM resistance and identify QTL, a diverse flax core collection of 372 accessions augmented with an additional 75 breeding lines were sequenced, and PM resistance was evaluated in the field for eight years (2010-2017) in Morden, Manitoba, Canada. Genome-wide association studies (GWAS) were performed using two single-locus and seven multi-locus statistical models with 247,160 single nucleotide polymorphisms (SNPs) and the phenotypes of the 447 individuals for each year separately as well as the means over years. A total of 349 quantitative trait nucleotides (QTNs) were identified, of which 44 large-effect QTNs (R2 = 10-30%) were highly stable over years. The total number of favourable alleles per accession was significantly correlated with PM resistance (r = 0.74), and genomic selection (GS) models using all identified QTNs generated significantly higher predictive ability (r = 0.93) than those constructed using the 247,160 genome-wide random SNP (r = 0.69), validating the overall reliability of the QTNs and showing the additivity of PM resistance in flax. The QTNs were clustered on the distal ends of all 15 chromosomes, especially on chromosome 5 (0.4-5.6 Mb and 9.4-16.9 Mb) and 13 (4.7-5.2 Mb). To identify candidate genes, a dataset of 3230 SNPs located in resistance gene analogues (RGAs) was used as input for GWAS, from which an additional 39 RGA-specific QTNs were identified. Overall, 269 QTN loci harboured 445 RGAs within the 200 Kb regions spanning the QTNs, including 45 QTNs located within the RGAs. These RGAs supported by significant QTN/SNP allele effects were mostly nucleotide binding site and leucine-rich repeat receptors (NLRs) belonging to either coiled-coil (CC) NLR (CNL) or toll interleukin-1 (TIR) NLR (TNL), receptor-like kinase (RLK), receptor-like protein kinase (RLP), transmembrane-coiled-coil (TM-CC), WRKY, and mildew locus O (MLO) genes. These results constitute an important genomic tool for resistance breeding and gene cloning for PM in flax.

Fungicide Management of Pasmo Disease of Flax and Sensitivity of Septoria linicola to Pyraclostrobin and Fluxapyroxad.

Among the diseases that have the potential to cause damage to flax (Linum usitatissimum L.) every year, the fungal disease pasmo, caused by Septoria linicola, is the most important. Fungicide application and a diverse crop rotation are the most important strategies to control this disease because there is little variation in resistance among flax cultivars. However, few fungicide products are available to flax growers. Field studies were conducted at four locations in Western Canada in 2014, 2015, and 2016 to determine the effect of two fungicide active ingredients applied singly and in combination: pyraclostrobin, fluxapyroxad, and fluxapyroxad + pyraclostrobin; and two application timings (early-flower, mid-flower, and at both stages) on pasmo disease severity, seed yield, and quality of flaxseed. The results indicated that among the three fungicide treatments, both pyraclostrobin and fluxapyroxad + pyraclostrobin controlled pasmo effectively; however, fluxapyroxad + pyraclostrobin was the most beneficial to improve the quality and quantity of the seed for most of the site-years. Disease severity in the fungicide-free control was 70%, and application of fluxapyroxad + pyraclostrobin decreased disease severity to 18%, followed by pyraclostrobin (23%) and fluxapyroxad (48%). Application of fluxapyroxad + pyraclostrobin also improved seed yield to 2,562 kg ha-1 compared with 1,874 kg ha-1 for the fungicide-free control, followed by pyraclostrobin (2,391 kg ha-1) and fluxapyroxad (2,340 kg ha-1). Fungicide application at early and mid-flowering stage had the same effects on disease severity and seed yield; however, seed quality was improved more when fungicide was applied at mid-flowering stage. Continuous use of the same fungicide may result in the development of fungicide insensitivity in the pathogen population. Thus, sensitivity of S. linicola isolates to pyraclostrobin and fluxapyroxad fungicides was determined by the spore germination and microtiter assay methods. Fungicide insensitivity was not detected among the 73 isolates of S. linicola tested against either of these fungicides.

Evaluation of Genomic Prediction for Pasmo Resistance in Flax.

Pasmo (Septoria linicola) is a fungal disease causing major losses in seed yield and quality and stem fibre quality in flax. Pasmo resistance (PR) is quantitative and has low heritability. To improve PR breeding efficiency, the accuracy of genomic prediction (GP) was evaluated using a diverse worldwide core collection of 370 accessions. Four marker sets, including three defined by 500, 134 and 67 previously identified quantitative trait loci (QTL) and one of 52,347 PR-correlated genome-wide single nucleotide polymorphisms, were used to build ridge regression best linear unbiased prediction (RR-BLUP) models using pasmo severity (PS) data collected from field experiments performed during five consecutive years. With five-fold random cross-validation, GP accuracy as high as 0.92 was obtained from the models using the 500 QTL when the average PS was used as the training dataset. GP accuracy increased with training population size, reaching values >0.9 with training population size greater than 185. Linear regression of the observed PS with the number of positive-effect QTL in accessions provided an alternative GP approach with an accuracy of 0.86. The results demonstrate the GP models based on marker information from all identified QTL and the 5-year PS average is highly effective for PR prediction.

Genome-Wide Association Study and Selection Signatures Detect Genomic Regions Associated with Seed Yield and Oil Quality in Flax.

A genome-wide association study (GWAS) was performed on a set of 260 lines which belong to three different bi-parental flax mapping populations. These lines were sequenced to an averaged genome coverage of 19× using the Illumina Hi-Seq platform. Phenotypic data for 11 seed yield and oil quality traits were collected in eight year/location environments. A total of 17,288 single nucleotide polymorphisms were identified, which explained more than 80% of the phenotypic variation for days to maturity (DTM), iodine value (IOD), palmitic (PAL), stearic, linoleic (LIO) and linolenic (LIN) acid contents. Twenty-three unique genomic regions associated with 33 quantitative trait loci (QTL) for the studied traits were detected, thereby validating four genomic regions previously identified. The 33 QTL explained 48⁻73% of the phenotypic variation for oil content, IOD, PAL, LIO and LIN but only 8⁻14% for plant height, DTM and seed yield. A genome-wide selective sweep scan for selection signatures detected 114 genomic regions that accounted for 7.82% of the flax pseudomolecule and overlapped with the 11 GWAS-detected genomic regions associated with 18 QTL for 11 traits. The results demonstrate the utility of GWAS combined with selection signatures for dissection of the genetic structure of traits and for pinpointing genomic regions for breeding improvement.

Identification and application of exogenous dsRNA confers plant protection against Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum, the causal agent of white stem rot, is responsible for significant losses in crop yields around the globe. While our understanding of S. sclerotiorum infection is becoming clearer, genetic control of the pathogen has been elusive and effective control of pathogen colonization using traditional broad-spectrum agro-chemical protocols are less effective than desired. In the current study, we developed species-specific RNA interference-based control treatments capable of reducing fungal infection. Development of a target identification pipeline using global RNA sequencing data for selection and application of double stranded RNA (dsRNA) molecules identified single gene targets of the fungus. Using this approach, we demonstrate the utility of this technology through foliar applications of dsRNAs to the leaf surface that significantly decreased fungal infection and S. sclerotiorum disease symptoms. Select target gene homologs were also tested in the closely related species, Botrytis cinerea, reducing lesion size and providing compelling evidence of the adaptability and flexibility of this technology in protecting plants against devastating fungal pathogens.

Genome-Wide Association Studies for Pasmo Resistance in Flax (Linum usitatissimum L.).

Pasmo is one of the most widespread diseases threatening flax production. To identify genetic regions associated with pasmo resistance (PR), a genome-wide association study was performed on 370 accessions from the flax core collection. Evaluation of pasmo severity was performed in the field from 2012 to 2016 in Morden, MB, Canada. Genotyping-by-sequencing has identified 258,873 single nucleotide polymorphisms (SNPs) distributed on all 15 flax chromosomes. Marker-trait associations were identified using ten different statistical models. A total of 692 unique quantitative trait nucleotides (QTNs) associated with 500 putative quantitative trait loci (QTL) were detected from six phenotypic PR datasets (five individual years and average across years). Different QTNs were identified with various statistical models and from individual PR datasets, indicative of the complementation between analytical methods and/or genotype × environment interactions of the QTL effects. The single-locus models tended to identify large-effect QTNs while the multi-loci models were able to detect QTNs with smaller effects. Among the putative QTL, 67 had large effects (3-23%), were stable across all datasets and explained 32-64% of the total variation for PR in the various datasets. Forty-five of these QTL spanned 85 resistance gene analogs including a large toll interleukin receptor, nucleotide-binding site, leucine-rich repeat (TNL) type gene cluster on chromosome 8. The number of QTL with positive-effect or favorite alleles (NPQTL) in accessions was significantly correlated with PR (R 2 = 0.55), suggesting that these QTL effects are mainly additive. NPQTL was also significantly associated with morphotype (R 2 = 0.52) and major QTL with positive effect alleles were present in the fiber type accessions. The 67 large effect QTL are suited for marker-assisted selection and the 500 QTL for effective genomic prediction in PR molecular breeding.

Genetic Variability of 27 Traits in a Core Collection of Flax (Linum usitatissimum L.).

Assessment of genetic variability of plant core germplasm is needed for efficient germplasm utilization in breeding improvement. A total of 391 accessions of a flax core collection, which preserves the variation present in the world collection of 3,378 accessions maintained by Plant Gene Resources of Canada (PGRC) and represents a broad range of geographical origins, different improvement statuses and two morphotypes, was evaluated in field trials in up to 8 year-location environments for 10 agronomic, eight seed quality, six fiber and three disease resistance traits. The large phenotypic variation in this subset was explained by morphotypes (22%), geographical origins (11%), and other variance components (67%). Both divergence and similarity between two basic morphotypes, namely oil or linseed and fiber types, were observed, whereby linseed accessions had greater thousand seed weight, seeds m-2, oil content, branching capability and resistance to powdery mildew while fiber accessions had greater straw weight, plant height, protein content and resistance to pasmo and fusarium wilt diseases, but they had similar performance in many traits and some of them shared common characteristics of fiber and linseed types. Weak geographical patterns within either fiber or linseed accessions were confirmed, but specific trait performance was identified in East Asia for fiber type, and South Asia and North America for linseed type. Relatively high broad-sense heritability was obtained for seed quality traits, followed by agronomic traits and resistance to powdery mildew and fusarium wilt. Diverse phenotypic and genetic variability in the flax core collection constitutes a useful resource for breeding.

Triploid Production from Interspecific Crosses of Two Diploid Perennial Helianthus with Diploid Cultivated Sunflower (Helianthus annuus L.).

Wild Helianthus species are a valuable genetic resource for the improvement of cultivated sunflower. We report the discovery and characterization of a unique high frequency production of triploids when cultivated sunflower was pollinated by specific accessions of diploid Helianthus nuttallii T. & G. and H. maximiliani Schr. Genomic in situ hybridization (GISH) analyses indicated that the triploid F1s had two genomes from the wild pollen sources and one from the cultivated line. Mitotic chromosome analyses indicated that the frequency of triploid progenies from the crosses of cultivated lines × H. nuttallii accession 102 (N102) was significantly higher than those of unexpected polyploid progenies from the crosses of wild perennial species × N102, and no unexpected polyploids were obtained from the reverse crosses. Pollen stainability analysis suggested the existence of a low percentage of unreduced (2n) male gametes in some accessions, especially N102 and H. maximiliani accession 1113 (M1113), which were generated at the telophase II and tetrad stages of meiosis. The triploid F1s could be the results of preferred fertilization of the low frequency of 2n male gametes with the female gametes of the cultivated sunflower, due to the dosage factors related to recognition and rejection of foreign pollen during fertilization. The triploids have been used to produce amphiploids and aneuploids. Future studies of the male gametes' fate from pollination through fertilization will further uncover the mechanism of this whole genome transmission. Studies of the genetic control of this trait will facilitate research on sunflower polyploidy speciation and evolution, and the utilization of this trait in sunflower breeding.

Identification of antifungal antibiotics of Bacillus species isolated from different microhabitats using polymerase chain reaction and MALDI-TOF mass spectrometry.

Although many Bacillus species are known to be good antibiotic producers capable of acting as biocontrol agents, the underlying antimicrobial mechanisms are often poorly understood. In this study, 21 Bacillus strains, demonstrating over 50% mycelial inhibition against Sclerotinia sclerotiorum as well as significant control in plant assays, were examined for the presence of antibiotic biosynthetic genes. Primers specific for bacillomycin D, iturin A, surfactin, mycosubtilin, fengycin, and zwittermicin A were used to amplify biosynthetic genes from these bacteria using PCR. The majority of strains harbored surfactin (21/21) and iturin A (20/21) biosynthetic genes. Three strains (Bacillus subtilis 3057, Bacillus amyloliquefaciens BS6, and Bacillus mycoides 4079) were positive for bacillomycin D, whereas 4 strains (B. subtilis H-08-02, B. subtilis 3057, B. amyloliquefaciens BS6, and B. mycoides 4079) showed the presence of the fengycin biosynthetic gene. The zwittermicin A gene was detected in B. mycoides S, Bacillus thuringiensis BS8, and B. amyloliquefaciens BS6. Sequence analysis of purified PCR products revealed homology with corresponding genes from other Bacillus sp. in the GenBank database. Production of particular antibiotics in strains BS6, H-08-02, 3057, and 4079 was confirmed through matrix-assisted laser desorption ionization - time of flight - mass spectroscopy (MALDI-TOF-MS). This study revealed the equivalent capability of different Bacillus strains from various microhabitats to produce the above-mentioned antibiotics and highlights the possibility of using some strains as potential biocontrol agents under different microhabitats distant from their original habitat. Furthermore, it will enable researchers to develop rational strategies for the application of the antagonists and their metabolites within an agroecosystem. To the best of our knowledge, this is the first report of a B. mycoides strain that carries biosynthetic genes and produces fengycin and surfactin.

A rust-inducible gene from flax (fis1) is involved in proline catabolism.

A gene fis1 from flax (Linum usitatissimum), which is induced in mesophyll cells at the site of rust (Melampsora lini) infection, is also expressed in vascular tissue, particularly in floral structures of healthy plants. This paper reports that the promoter controlling this expression is contained within 282 bp 5' to the coding region and that fis1 gene induction is specifically by the rust pathogen and not by other fungal pathogens or by wounding. The fis1 gene has 73% homology with an Arabidopsis gene which encodes delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH) which is a part of the proline degradation pathway. Transgenic flax plants that either over-express fis1 or show reduced fis1 expression due to RNA-mediated gene silencing have an unaltered morphology. However, plants with reduced fis1 expression have markedly increased sensitivity to exogenous proline and show alteration in epidermal cell morphology, callose deposition and the production of hydrogen peroxide during proline-induced death. These lines, which show a biologically significant level of fis1 suppression, have an unaltered reaction to either virulent or avirulent rust infections, as do fis1 over-expression lines. These data indicate that the fis1 gene plays a role in proline metabolism and most likely encodes for a P5CDH enzyme. However, the precise role of fis1 and P5C catabolism in the development of rust disease remains unclear.